Rotaviruses are ubiquitous agents that cause diarrheal disease in the young of not only humans but also many animal species. Several candidate vaccines against human rotavirus (HRV) disease are currently being evaluated with special emphasis on the VP7 outer capsid protein, which is a major protective antigen. Currently, eight different serotypes have been established among HRVs based on the antigenic specificity of VP7. The outer capsid protein, VP4, is also a major protective antigen, but relatively little is known about the antigenic relationships and extent of polymorphism of the VP4 of various HRVs. In previous studies, it was shown by sequence analysis that at least four alleles of the VP4 gene are present among HRVs. One allele of VP4 is present on strains recovered from patients with symptomatic infection. These strains had been classified as VP7 serotype 1 (strains KU and Wa), VP7 serotype 3 (strain P), or VP7 serotype 4 (strain VA70). These rotavirus strains, designated as VP4 genetic group 1, possess a highly conserved VP4 protein that exhibits 93-97% amino acid identity. A second allele of VP4 is present in HRVs belonging to VP7 serotype 2(strains DS-1 and RV-5) the exhibit 98% VP4 amino acid identity. These strains, designated as VP4 genetic group 2, were also recovered from symptomatic infections. Moreover, the VP4s of genetic group 2 are most closely related to the corresponding proteins of group 1, exhibiting an 89- 90% amino acid homology. A third allele of VP4 is present in HRV strains that were recovered from asymptomatic neonatal infections and that belong to VP7 serotype 1 (M37), VP7 serotype 2 (1076), VP7 serotype 3 (McN), or VP7 serotype 4 (ST3). the VP4s of these strains (designated VP4 genetic group 3) are highly related, as indicated by 95-97% amino acid identity. The VP4s of the third genetic group exhibit only a 75% amino acid identity with the corresponding VP4 proteins of groups 1 and 2. A fourth allele is present on the HRV K8 strain (belonging to VP7 serotype 1) that appears to have a unique VP4 gene exhibiting only 64-65% amino acid identity with the VP4 proteins of viruses of VP4 genetic group 1, 2, or 3. In a recent study, four different VP4 alleles present on HRV strains KU (genetic group 1), DS-1 (genetic group 2), 1076 (genetic group 3), and K8 (genetic group 4) were separately expressed in Spodoptera frugiperda (Sf9) cells using the baculovirus vector Autographa californica nuclear polyhedrosis virus. Antisera produced in response to immunization with these recombinant VP4 proteins allowed us to define the extent and distribution of VP4 antigenic polymorphism among rotaviruses currently circulating in humans.